When analyzing post-translational modifications, sequence variants, disulfide bonds, or any other variable modifications on peptides, valuable information in terms of the confidence in localization of the modification on the peptide sequence is captured in the Delta Mod. Score value. By definition, Delta Mod. Score is “the difference between the top-scoring match in an MSMS spectrum, and the next-best match that differs in any way (e.g., modification localization).” In practical terms, when a peptide has two possible modification sites, Delta Mod. Score shows whether the localization of the modification on the determined site is confident (high value) or not (low). The confidence is determined by the presence of MSMS fragment ions between two possible sites.
To give an example, peptide FNWYVDGVEVHNAK has two Asparagine residues, hence two possible locations for a deamidation variable modification. In the below MSMS ID and spectrum, Byonic shows deamidation on the first N: FN[+1]WYVDGVEVHNAK.
By looking at the Delta Mod. Score value, which ranges from 0 to the Score value of the peptide, we see that it is equal to the Score value, the maximum it can be. This indicates a very confident localization of the modification. In the above spectrum, there is strong evidence that the first N is deamidated, and not the second N. This is due to the fragment evidence in the spectrum between the two sites, namely y4 to y12 and b3 to b11 series.
In contrast, the below example shows another peptide with 3 Asparagine residues. The MSMS ID in this case has a Delta Mod. Score of 0.
Although there are fragment ions between the 1stand the 2ndN, there are no ions between the 2ndand the 3rd. Hence the modification location has no confidence; the deamidation can be on the 2ndor the 3rdN, there is no way to tell with the evidence present in this spectrum. It should be noted that this doesn’t indicate a false positive ID. There is no question that the peptide is deamidated, only that the deamidation location on the peptide sequence is ambiguous.
Delta Mod Score is an extremely useful value in variant analyses, particularly in peptides with multiple possible modification positions. It is available in Byos Peptide workflows, Byonic-Viewer, and Byologic projects.